Nonthermal excitation effects mediated by sub-terahertz radiation on hydrogen exchange in ubiquitin

Abstract

Water dynamics in the hydration layers of biomolecules play crucial roles in a wide range of biological functions. A hydrated protein contains multiple components of diffusional and vibrational dynamics of water and protein, which may be coupled at ∼0.1-THz frequency (10-ps timescale) at room temperature. However, the microscopic description of biomolecular functions based on various modes of protein-water-coupled motions remains elusive. A novel approach for perturbing the hydration dynamics in the subterahertz frequency range and probing them at the atomic level is therefore warranted. In this study, we investigated the effect of klystron-based, intense 0.1-THz excitation on the slow dynamics of ubiquitin using NMR-based measurements of hydrogen-deuterium exchange. We demonstrated that the subterahertz irradiation accelerated the hydrogen-deuterium exchange of the amides located in the interior of the protein and hydrophobic surfaces while decelerating this exchange in the amides located in the surface loop and short 3₁₀ helix regions. This subterahertz-radiation-induced effect was qualitatively contradictory to the increased-temperature-induced effect. Our results suggest that the heterogeneous water dynamics occurring at the protein-water interface include components that are nonthermally excited by the subterahertz radiation. Such subterahertz-excited components may be linked to the slow function-related dynamics of the protein.

Figure 1

Schematic representation of subterahertz radiation experimental setup. (A and B) Subterahertz irradiation (A) and subterahertz-dependent heat conduction (B) to ubiquitin (Ub) solution using the same klystron-based subterahertz source. Devices for subterahertz radiation were connected through rectangular waveguides, WR-10. The length unit is shown in millimeter. See Materials and methods for details. (C) THz-HDX experiment. Lyophilized Ub was dissolved in 100 μL D₂O to obtain 0.5 mM Ub solution, which was thoroughly mixed using a vortex mixer and was then injected into the sample cell within 1 min at room temperature (rt) of ∼25°C. The sample was incubated for 13 min at rt and was subjected to temperature increase (+ΔT) caused by subterahertz radiation or heat conduction for a variable time (X = 0, 3, 6, or 12 min). The experiment conducted at X = 0 corresponds to control. After 14 min of dissolution in D₂O, Ub solution was collected in the sample tube and stored at 4°C until NMR-based HDX measurement was performed. The time interval between the sample collection and NMR measurement per condition is shown in Fig. S2.

Figure 2

Effect of subterahertz irradiation at low (left) and high (middle) power density and temperature increase (right) on the amide proton exchange of Ub. The tertiary Ub structures with 180° rotation are shown (Protein Data Bank, PDB: 1UBQ) (42). Amide nitrogen atoms of the analyzed residues are shown (see main text for details). Amino acid residues were mapped in the Ub structure when the HDX of the main chain amide groups was accelerated or decelerated. An example of L69 is shown in an inset. The acceleration or deceleration of HDX in each residue (schematically indicated with an arrow) was defined using the signal intensity ratio of each measurement to that of GC (I/I_(GC); see Materials and methods for details). When I/I_(GC) was decreased or increased (i.e., HDX was accelerated or decelerated) after subterahertz radiation or heating above the measurement error range, the corresponding residue was colored orange or blue, respectively. Dark and light colors indicate that the acceleration or deceleration of HDX was detected at 6 and 12 min and only at 12 min, respectively. Note that 3 min was selected as the reference time point at which the sample temperature reached plateau after subterahertz irradiation or heating. Error bars are derived from signal/noise ratio, following Eq. 6.